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Marine Biotechnology

, Volume 8, Issue 5, pp 560–566 | Cite as

Variants of Green Fluorescent Protein GFPxm

  • Wen-Xin Luo
  • Tong Cheng
  • Bao-Quan Guan
  • Shao-Wei Li
  • Ji Miao
  • Jun Zhang
  • Ning-Shao Xia
Original Article

Abstract

As research progresses, fluorescent proteins useful for optical marking will evolve toward brighter, monomeric forms that are more diverse in color. We previously reported a new fluorescent protein from Aequorea macrodactyla, GFPxm, that exhibited many characteristics similar to wild-type green fluorescent protein (GFP). However, the application of GFPxm was limited because GFPxm expressed and produced fluorescence only at low temperatures. To improve the fluorescent properties of GFPxm, 12 variants were produced by site-directed mutagenesis and DNA shuffling. Seven of these mutants could produce strong fluorescence when expressed at 37°C. The relative fluorescence intensities of mutants GFPxm16, GFPxm18, and GFPxm19 were higher than that of EGFP (enhanced GFP) when the expression temperature was between 25 and 37°C, and mutants GFPxm16 and GFPxm163 could maintain a high fluorescence intensity even when expressed at 42°C. Meanwhile, at least 4 mutants could be successfully expressed in mammalian cell lines. The fluorescence spectra of 6 of the 12 mutants had a progressive red shift. The longest excitation-emission maximum was at 514/525 nm. In addition, 3 of the 12 mutants had two excitation peaks including an UV-excitation peak, while another mutant had only one UV-excitation peak.

Keywords

Fluorescence spectra green fluorescent protein variants 

References

  1. Ando, R, Hama, H, Yamamoto-Hino, M, Mizuno, H, Miyawaki, A 2002An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent proteinProc Natl Acad Sci USA991265112656CrossRefGoogle Scholar
  2. Ando, R, Mizuno, H, Miyawaki, A 2004Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlightingScience30613701373CrossRefGoogle Scholar
  3. Campbell, RE, Tour, O, Palmer, AE, Steinbach, PA, Baird, GS, Zacharias, DA, Tsien, RY 2002A monomeric red fluorescent proteinProc Natl Acad Sci USA9978777882CrossRefGoogle Scholar
  4. Chudakov, DM, Belousov, VV, Zaraisky, AG, Novoselov, VV, Staroverov, DB, Zorov, DB, Lukyanov, S, Lukyanov, KA 2003Kindling fluorescent proteins for precise in vivo photolabelingNat Biotechnol21191194CrossRefGoogle Scholar
  5. Chudakov, DM, Verkhusha, VV, Staroverov, DB, Souslova, EA, Lukyanov, S, Lukyanov, KA 2004Photoswitchable cyan fluorescent protein for protein trackingNat Biotechnol2214351439CrossRefGoogle Scholar
  6. Crameri, A, Whitehorn, EA, Tate, E, Stemmer, WPC 1996Improved green fluorescent protein by molecular evolution using DNA shufflingNat Biotechnol14315319CrossRefGoogle Scholar
  7. Cubitt, AB, Woollenweber, LA, Heim, R 1999Understanding structure-function relationships in the Aequorea victoria green fluorescent proteinMethods Cell Biol581930CrossRefGoogle Scholar
  8. Gurskaya, NG, Fradkov, AF, Pounkova, NI, Staroverov, DB, Bulina, ME, Yanushevich, YG, Labas, YA, Lukyanov, S, Lukyanov, KA 2003A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutantsBiochem J373403408CrossRefGoogle Scholar
  9. Ito, Y, Suzuki, M, Husimi, Y 1999A novel mutant of green florescent protein with enhanced sensitivity for microanalysis at 488 nm excitationBiochem Biophys Res Commun264556560CrossRefGoogle Scholar
  10. Karasawa, S, Araki, T, Yamamoto-Hino, M, Miyawaki, A 2003A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labelingJ Biol Chem2783416734171CrossRefGoogle Scholar
  11. Labas, YA, Gurskaya, NG, Yanushevich, YG, Fradkov, AF, Lukyanov, KA, Lukyanov, SA, Matz, MV 2002Diversity and evolution of the green fluorescent protein familyProc Natl Acad Sci USA9942564261CrossRefGoogle Scholar
  12. Lippincott-Schwartz, J, Patterson, GH 2003Development and use of fluorescent protein markers in living cellsScience3008791CrossRefGoogle Scholar
  13. Luo, WX, Zhang, J, Yang, HJ, Li, SW, Xie, XY, Pang, SQ, Li, SJ, Xia, NS 2000Construction and application of an Escherichia coli high effective expression vector with an enhancerSheng Wu Gong Cheng Xue Bao16578581Google Scholar
  14. Ormo, M, Cubitt, AB, Kallio, K, Gross, LA, Tsien, RY, Remington, SJ 1996Crystal structure of the Aequorea victoria green fluorescent proteinScience27313921395Google Scholar
  15. Patterson, GH, Lippincott-Schwartz, J 2002A photoactivatable GFP for selective photolabeling of proteins and cellsScience29718731877CrossRefGoogle Scholar
  16. Shaner, NC, Campbell, RE, Steinbach, PA, Giepmans, BN, Palmer, AE, Tsien, RY 2004Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent proteinNat Biotechnol2215671572CrossRefGoogle Scholar
  17. Tsien, RY 1998The green fluorescent proteinAnnu Rev Biochem67509544CrossRefGoogle Scholar
  18. Verkhusha, VV, Sorkin, A 2005Conversion of the monomeric red fluorescent protein into a photoactivatable probeChem Biol12279285CrossRefGoogle Scholar
  19. Xia, NS, Luo, WX, Zhang, J, Xie, XY, Yang, HJ, Li, SW, Chen, M, Ng, MH 2002Bioluminescence of Aequorea macrodactyla, a common jellyfish species found in the east China seaMar Biotechnol4155162CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Wen-Xin Luo
    • 1
  • Tong Cheng
    • 1
  • Bao-Quan Guan
    • 1
  • Shao-Wei Li
    • 1
  • Ji Miao
    • 1
  • Jun Zhang
    • 1
  • Ning-Shao Xia
    • 1
  1. 1.National Institute of Diagnostics and Vaccine Development in infectious disease, The Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell EngineeringXiamen UniversityXiamenPeoples’ Republic of China

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